Project Summary R-loops are three-stranded nucleic acid structures that consist of an RNA-DNA hybrid and a displaced single-stranded DNA. They are found in a broad range of organisms, from bacteria to humans, and play roles in gene expression regulation, DNA and histone modification, generating antibody diversity, and DNA repair. Conversely, R-loops can also be a source of genomic instability, and they have been associated with various neurological diseases and cancers. Given this dichotomy, a clear understanding of R-loop formation and resolution is essential to comprehend how they play both healthy and harmful roles in cellular function. Although mounting evidence suggests that R-loop formation and resolution depend on torsion in the DNA molecule, the mechanics of this interaction are poorly understood due to a lack of appropriate investigation methodologies. A correlation between transcriptional pausing and R- loop formation has also been reported, however the transient nature of RNA-DNA hybrids complicate analysis of these dynamic interactions. Single-molecule studies are well-suited to investigate both R-loop formation and the effects of R-loops on transcription because they permit direct observation of dynamic processes. The goal of the proposed research is to elucidate R-loop dynamics and investigate their impact on transcription. This will be accomplished through two aims. In Aim 1, the role of torsion in co-transcriptional R-loop formation will be identified. A combination of magnetic tweezers and single-molecule fluorescence techniques will be used to directly visualize the presence of RNA-DNA hybridization during transcription and investigate the relationship between negative DNA supercoiling and R-loop formation. In Aim 2, the impact of R-loops on transcription will be characterized. R-loop hybridization will be directly visualized and the progression of RNA polymerase will be tracked, in real time, to investigate how R-loop formation may impact gene expression. This work will address fundamental gaps in knowledge concerning R-loop dynamics, and will help to clarify the roles that R-loops play in essential cellular processes such as transcription and gene expression. This essential information will help to define the molecular origin of a variety of human diseases that are connected with genetic instability, and will provide a foundation upon which to develop novel preventative and therapeutic treatments.